Myxococcus xanthus is a unicellular prokaryote which forms multicellular structures containing heat-resistant, differentiated spores when starved for nutrients. We propose to study MglA, a GTP-binding protein required for both the morphogenesis of this multicellular structure (a fruiting body) and the differentiation of spores but not for vegetative growth. M. xanthus is the only known prokaryote which has a protein in the family of small (20-25kDa) molecular weight ras-like G-proteins. Like its homolog, H-ras, mutations that destroy the consensus GTP-binding region of mglA abolish its activities. However, unlike all other eukaryotic G-proteins in this family, MglA is not essential. In addition to affecting morphogenesis and sporogenesis, mglA- mutations also abolish motility. Our results show that MglA interacts with MglB, the product of the co-expressed gene in its transcription unit. We have found that mglB-lacZ translational fusions block motility during development but not during growth, suggesting that MglB-MglA interactions are different during growth and development. Experiments outlined in this proposal are aimed at exploring these interactions biochemically. The mechanism of regulation by mgl is complex. mgl- mutations affect expression during development of at least four genes, defined by insertions of Tn5-lac, which may explain why mgl- mutants fail to form fruiting bodies and spores. In addition, the mglA gene is required for activation of its own expression during growth and repression of its own expression during development. Identification of the cis and trans-acting elements required for autoregulation of mgl will provide new insights into G-protein signaling. We have discovered that the sporulation defect of mglA- mutants can be rescued by SAR1, a yeast GTP-binding protein in the ras family. Hence, the inability of mglA- mutants to form heat-resistant spores cannot completely be explained by their motility defect, because SAR1 does not restore motility. It takes a second-site mutation to revert the motility defect of an M. xanthus mglA- SAR1 strain. Our proposed analysis of mutations which restore motility to this strain will provide new insights into G-protein function.